TW200522973A - Neutralization agent of vacuolization toxin - Google Patents

Abstract

It is intended to provide a drug, a quasi drug, a food or a drink having an effect of preventing, preventing recurrence or treating digestive diseases, in which Helicobacter pylori participates, as well as an effect of killing Helicobacter pylori while showing little side effects and being free from any risk of the occurrence of a tolerant strain. Namely, a drug, a quasi drug, a food or a drink containing, as the active ingredient, proanthocyanidines having an effect of neutralizing (attenuating) a cavitating toxin produced by Helicobacter pylori, particulaly preferably proanthocyanigines originating in immature apple fruit or hop bract.

Description

200522973 IX. Description of the invention: [Technical field to which the invention belongs] The present invention relates to grape seed proanthocyanidins having the effect of detoxifying the cytosolic toxins produced by Helicobacter pylori, and in particular, it contains 5 Apple, or grape seed proanthocyanidins of Hubuhua as active ingredients, pharmaceuticals for the prevention, prevention of recurrence or treatment of Helicobacter pylori-related digestive organ diseases, quasi-drugs and food and beverages, and Helicobacter pylori Neutralizers of cytotoxic toxins, pharmaceuticals, quasi-drugs and food and beverages.

[Prior art] 10 Hubu flower is a perennial plant of the genus Hops, and its blanket flower (mature unfertilized female flower) is generally called Hubu flower. In addition to its flower part, there are various parts such as leaves, vines, and roots. The hubusu part (yellow particles formed in the root of the inner bud of the blanket fruit) in the hubuhua flower is the bitter and aromatic body of hubuhua, which is juxtaposed with yeast and malt in the brewing of beer It is an important beer ingredient.

15 In addition, hubuhua is commonly used in folk remedies as a sedative or anti-prostitution agent. The hubu flower bud removes the hubusu part from the hubu flower. It is not useful for beer brewing. Sometimes it is removed during beer brewing and formed as a by-product. At this time, Hubu buds have not been found to have other particularly effective uses other than fertilizers for soil improvement. Therefore, it is expected to develop use methods with higher added value. 20 Furthermore, in Patent Documents 1, 2, 3, 4, 5, and 6 related to the applicant's application, it was confirmed that polyphenols derived from Khubu flower, especially Khubu flower bud, have an antioxidant effect. 5. Stabilizing effect on foaming malt beverage, anti-corrosion effect, deodorizing effect, inhibiting cancer cell metastasis effect, DNA-Topoisomerase blocking effect. Moreover, in Patent Document 5, 200522973, it is recognized that it has a protein toxin effect for neutralizing RNAN-glycoside hydrazone activity or adp ribosyltransferase (ADP-ribosyltransferase) activity. However, as far as the anthocyanins of grape seed pupa from Khubuhua are concerned, no examples have so far revealed that they have the effect of neutralizing (non-toxicizing) the cytosolic toxin produced by Helicobacter pylori. Helicobacter pylori is a Gram-negative bacterium with a spiral form. Since Valian and Marshall reported their existence (Non-Patent Document 1), it has been known to be related to the pathogenesis of digestive organ diseases such as acute gastritis, chronic gastritis, gastric ulcer, and duodenal ulcer. They are closely related (see Non-Patent Documents 2, 3, and 4). Moreover, from the point that more than 90% of 10 patients with gastric cancer are carriers of Helicobacter pylori, H. pylori is highly likely to be associated with gastric cancer. WH0 even published in 1994 that "Helicobacter pylori Carcinogenic factors of gastric cancer. " The pathogenic factors produced by Helicobacter pylori have so far reported urease, hydrogen peroxide decomposing tritium, lipopolysaccharide (LPS), etc. In recent years, single administration on 15 mucosal cells of the stomach can cause cavitation degeneration of cavitation. As a result, toxin (VacA) was confirmed to cause gastritis in animal models (Non-Patent Document 5). Therefore, the understanding of cytosolic toxin as the main etiological factor of Helicobacter pylori has increased rapidly. Since then, for the treatment of ulcerative diseases such as gastric ulcer and duodenal ulcer, we have used anti-ulcer agents such as Sofa_, Pronoto, and proton pump inhibitors (PPI) such as omeprazole and lansoprazole 20 ); Famotidine, Simitidine and other gastric acid secretion inhibitors (H2 blockers) and so on. However, these drugs do not have the effect of inhibiting the proliferation of Helicobacter pylori, but are symptomatic therapeutic agents for ulcerative diseases. Therefore, even if the above-mentioned drugs are used to cure ulcerative diseases, H. pylori still remains in the body, so the recurrence rate within one year after the treatment is as high as 80-90%. 200522973 In order to overcome the above disadvantages, in addition to symptomatic therapy, In addition, there is also a treatment method for sterilizing Helicobacter pylori, that is, clinically using antibiotics such as amoxicillin, clarithromycin, nitropyrazole ethanol, tinidazole, which have antibacterial effect on Helicobacter pylori. At present, the combination of positron pump blocker and antibiotic 2 is the so-called new 53 combination therapy is the mainstream of sterilization treatment.

However, the new three-dose combination therapy must be administered in a large amount for a long period of time, so clinical problems such as the side effects of medicines or the occurrence of microbial alternations have actually occurred. In addition, the use of antibiotics may cause the cavitation toxins of causative factors produced by Helicobacter pylori to be excreted in large quantities around the gastric mucosa with the destruction of the bacteria. In addition, the large use of antibiotics may lead to the production of new and more tenacious financial bacteria. Based on the above knowledge, it is difficult to consider the new three-dose combination therapy that is widely used as an ideal treatment.

In Japan, Helicobacter pylori infection is particularly high in generations over 40 years of age, and the incidence of ulcerative diseases and gastric cancer is also higher than in Europe and the United States. If you can find 15 treatments without side effects or bacterial resistance problems The value in the industry is great. [Patent Document 1] JP 09-002917 [Patent Document 2] JP 09-163969 [Patent Document 3] JP 09-295944 20 [Patent Document 4] JP 10-025232 [ Patent Document 5] JP 2000-327582 [Patent Document 6] JP 2001-039886 [Patent Document 7] International Publication No. 02/07826 [Non-Patent Document 1] Lancet, 1273-1275, (1983 ) 7 200522973 [Non-Patent Document 2] Med. J. Aust ·, 142,436 (1985) [Non-Patent Document 3] Gastroenterology, 102, 1575 (1992) [Non-Patent Document 4] N. Engl. Med., 328, 308 (1993 ) [Non-Patent Document 5] Infect · Immun. 63, 4154-4160 (1995) 5 [Summary of the Invention] Therefore, the object of the present invention is to provide prevention of Helicobacter pylori-related gastrointestinal diseases with few side effects, non-resistant bacteria, and prevention , Medicines for preventing recurrence or treatment, and neutrophilic toxins produced by Helicobacter pylori that have a neutralizing effect; 10 In view of the above situation, the present inventors tried to solve the problem by finding a factor that detoxifies the cytosolic toxin produced by Helicobacter pylori, rather than killing Helicobacter pylori. If the effective non-toxicizing factor of cytosolic toxin is found, its significance in the medical industry is incalculable. As a result of repeated studies, the present inventors have found that one of the 15 polyphosphates present in Hobflower and Apple can effectively detoxify the cavitation toxin produced by Helicobacter pylori, and thus completed the present invention. The immature fruit of the apple or the bud portion of the hubu flower contains the polyphenol particularly in a large amount. The polyphenol contained in hubuhua will be adsorbed on resins that show affinity with polyphenols such as acetophenone-diethyltoluene resin, and when treated with an ultrafiltration membrane with a molecular weight of 1,000 or more. It has the property of being impermeable to the membrane, and even when heated in an alcohol solution containing 20% 5% hydrochloric acid, it will be hydrolyzed to produce anthocyanins, so it is judged as grape seed proanthocyanidins. In addition, in the grape seed proanthocyanidins, in the GPC (glia transmission color analysis) analysis, the chromatogram as shown in FIG. 1 is given, and on the other hand, in the extinction analysis, the extinction shown in FIG. 2 is given. distributed. Also, the polyphenol contained in apples will also be adsorbed on styrene-divinylbenzene resin and the like, and it is added to an alcohol solution containing 5% hydrochloric acid. Therefore, it is judged that it is grape seed anthocyanin and s does not show affinity. When the protection of sex is hot, it will be hydrolyzed to produce ^ main element types, that is, the I + 5 of the present invention has neutralization from grapes or anthocyanins, especially chemical toxins. Agent. Anthracin is an active ingredient of cytotoxin, which is an active ingredient, 5,4-_2- (3-phenylalanylamino) benzyl I or phloretin, and even 4 Α β cut I phenols. Inhibits changes in the current on the cell membrane produced by cytotoxic toxins, 4 see ... accounted for by Tombola (Tombola F. et 10 15

al., FEBS Lett. 543, 18 M _ / 03. However, the same document also describes that 'the substances that inhibit the change in the current on the cell membrane in these systems have nothing to do with blocking intracellular cavitation or neutralizing cytotoxicity caused by cytosolic toxins. Also, 'the compounds disclosed in these documents as substances that inhibit changes in the current on the cell membrane', although they are polyphanes, are not compounds of grape seed proanthocyanidins. Therefore, the use of compounds derived from plants, especially from There have been no reports on the technology of detoxification of cavitation toxins by cloth flowers or apple grape anthocyanins. Effects of the Invention 20 The cavitation toxin neutralizing agent of the present invention has an effect of detoxifying the cavitation toxin, and is therefore effective in preventing and treating infectious diseases in which the cavitation toxin is a causative agent. The product of the present invention can be used as a preventive / therapeutic agent for infectious diseases in which cavitation toxins are the causative agents, and reagents for biochemical experiments, etc., and make them into products. Helicobacter pylori-related diseases of the digestive organs can include gastric ulcer, duodenal ulcer 200522973 ulcer, gastritis, gastric cancer, malt lymphoma and the like. [Embodiment] The raw material of the cytotoxic toxin neutralizer of the present invention is preferably an immature fruit of an apple. In addition, there is a vine flower or bud part of the flower. In particular, the apple or the flower part of the flower 5 can be used without separation. overall.

The so-called Khubu flower buds are obtained by removing the Khubusu part from the Khubu flower capsule. Generally speaking, after the Khubu flower capsule is crushed, the Khubusu part is removed by sieving to obtain the Khubusu. Cloth buds. However, recently, in beer brewing, in order to save the labor of sieving and removing the hubu buds, it tends to not remove the hubu buds that are useless on the 10 brewed beer, but directly shape the hubu buds into small ones. Spherical, used as a brewing ball for hubuhua. Therefore, the raw material of the present invention is not particularly limited as long as it includes the vine buds or buds of Khubu flowers, and the Khubu flower blanket fruit or Khubu flower pellets including Khubu flowers are not a problem.

The manufacturing method for obtaining the cytosolic toxin neutralizing agent from Hubuhua is Hubu15 flower vine, bud, or Hubu flower blanket fruit or Hubu flower ball containing Hubu flower bud, or contains the aforementioned Hubu flower The part of the value object is used as a raw material, and it is extracted with water or an aqueous solution of an organic solvent mixed with water, such as alcohol, ketone, acetonitrile and the like below 80v / v%. A suitable example is hydrous ethanol with ethanol below 50 v / v%. The ratio of the raw material to the extraction solvent is preferably 1: 20 to 100 (weight ratio). The extraction is performed at 4 to 95 ° C for 20 to 60 minutes with stirring. The extract can be obtained by filtration, but if necessary, filtration aids such as perlite can also be used. From the extract thus obtained, the solvent can be removed by a general method such as concentration, freeze-drying, and spray-drying to obtain a cavitation toxin as a powder. The cytotoxic toxin neutralizer obtained here can be fully used in practice, and if necessary, it can be further improved by the following method using the adsorption resin 10 200522973. However, this process is purely a step to improve the refined system and can be omitted if not necessary. The above extraction solution is treated with a granular synthetic resin having an affinity for polyphenols to concentrate the cavitation toxin neutralizing agent. In this step, the method can be performed by passing the Futaba extract solution through a column filled with the granular synthetic resin, washing the column sufficiently, and dissolving the cytosolic toxin neutralizer adsorbed on the column, or The granular resin was immersed in the hubuhua extract and was processed in a batch process. When the cavitation toxin is adsorbed on the synthetic resin, after cooling the hubuhua extract to room temperature of 15 ~ 30 C, if necessary, reduce the concentration of the organic solvent of the 10 extract in advance by decompression and concentration, etc. to increase Adsorption efficiency. As the material of the synthetic adsorbent, hydroxypropylated dextrin, hydrophilic ethylene polymer, and styrene-divinylbenzene polymer can be used. Then, the synthetic resin can be washed to further improve the fine system of the cytotoxic toxin neutralizer. The solvent used for washing is preferably water or even a biOw / w% ethanol aqueous solution 15, and washing with a solvent amount of 1 to 10 times the amount of resin is preferred. Next, from the synthetic resin adsorbing polyphenols, the cytosolic toxin neutralizer is removed and dissolved. As the solvent used for the dissolution, an aqueous alcohol, an aqueous ketone, an aqueous acetonitrile, or the like can be used. Particularly suitable examples include an aqueous ethanol solution of 30 w / w% or more. The amount of solvent passing through is preferably 2 to 6 times the amount of resin. 20 The general method such as concentration, freeze-drying, and spray-drying is used to remove the solvent from the obtained eluate to obtain a cavitation toxin neutralizing agent as a powder. When concentrated under reduced pressure, alcohol, ketone, acetonitrile and the like can be recovered and reused. The synthetic resin used can be reused after being washed with an alcohol aqueous solution of 80 Wv% or more, and a 0.05N aqueous sodium hydride solution. 200522973 The vaccinated toxin neutralizer obtained in this way can be directly used for practical use, or it can be further improved by using the method of crossing the membrane as described below. However, this process is purely a step to improve the refined system and can be omitted if not necessary. The hollow cell toxin neutralizing agent obtained by the above method is dissolved in water, or an organic solvent with water and 5 in order to isolate the ultra-transmembrane treatment material with a molecular weight of _0 or more. Cellulose, ethyl cellulose, and cellulose can be used. Acrylic, polyester, polyisocyanate, PVD, etc. are generally used as materials for ultrafiltration membranes, and there are no particular restrictions. In addition, the isolated molecular weight can be used as long as it is 10,000 or more, and there is no special problem. However, if a film with an excessively large isolated molecular weight is used, the yield will be greatly reduced, or the isolated molecular will be less than 10 hours, which will require time for processing. It becomes longer, so it is better to separate the ultrafiltration membrane with 5,000 ~ 50,000. The treatment depends on the type of the extraction solvent or the ratio of the extraction solvent to the hubu flower or hubu flower buds, but it is best to carry out until the amount of the upper residual liquid is about 1/10 to 1/100 at the beginning of the treatment. . The pressure at this time depends on the ultrafiltration membrane or the filtering device, but it is preferably about 15. In addition, if necessary, the once-processed upper residual liquid can be diluted again with an appropriate solvent such as water, and then processed in the same way to improve the precision system. The solvent of the obtained upper residual liquid can be removed by general methods such as concentration, freeze-drying, spray-drying, etc., to obtain a cavitated toxin as a powder. When concentrated under reduced pressure, alcohol, ketone, acetonitrile and the like can be recovered and reused. 20 The thus obtained cavitation toxin neutralizer is a slightly bitter, airless, tan, brown or even yellowish powder, which will be adsorbed on a synthetic resin with affinity for polyphenols, and will be isolated by ultrafiltration with a molecular weight of 1,000 or more. Grape seed proanthocyanidins that do not penetrate the membrane during membrane treatment. In addition, the yield side ® is 0.5 to 20 0 w / w% when converted to the weight of Hubuhua dad, and 200522973 is 0.5 to 15.0w / w% when converted to the weight of Hubuhua capsule. The manufacturing method when obtaining a cavitation toxin neutralizing agent from apples is to squeeze the juice from apple fruit, especially an immature apple, to obtain a solution containing a cavitation toxin neutralizing agent, and then concentrate the solution and freeze-dry , Spray drying and other methods to make powder for use in general method. In addition, if necessary, a column filled with a granular resin having affinity for polyphenols and the like can be used to purify the cytosolic toxin to improve the degree of purification. This step is the same as the concentration and purification step operation of the cytosolic toxin neutralizer obtained from Hobflower.

The cytotoxic toxin neutralizer obtained in this way can be formulated with commonly used carriers, 10 auxiliaries, additives, etc., and orally or non-oral products are used as pharmaceuticals according to common methods, or mixed with food materials. And as food and drink.

Oral preparations for pharmaceuticals include lozenges, capsules, granules, syrups, etc., while non-oral preparations include external preparations such as ointments, creams, and water, injections such as sterile solutions or suspensions. When these products are administered to the human body by medicine, the dosage of 15 2 mg to 500 mg is administered 1 to several times a day, that is, the total dosage is 2 mg to 1000 mg, and the effect can be fully achieved. The medicinal product containing a neutrophil toxin neutralizing agent of the present invention can obtain the required unit capacity form with physiologically acceptable vehicles, carriers, excipients, blending agents, stabilizers, flavoring agents, and the like. The adjuvants mixed in the bonding agent and capsule are as follows. Such as the tragacanth gum, acacia gum, corn starch, gelatin binding agent; such as excipients of microcrystalline cellulose; such as corn starch, fully gelatinized starch, arginine bulking agent; such as magnesium stearate Smoothing agent; such as sweetener of sucrose, lactose, saccharin; flavoring agent of mint, red pearl oil, cherry, etc. In addition, in the case of capsules, in addition to the above-mentioned materials, liquid carriers such as oils and fats can be contained. In addition, other materials can be coated with 13 200522973, or the physical form of the preparation can be changed by other methods. For example, the bonding agent may be coated with shellac or sugar. Syrups or St can contain granulated sugar as a sweetened pair of mesitynic acid " or p-benzoic acid-as a flavoring agent that is anti-smell, rosy, or willow-like. 5 10 15

Sterile compositions used as injectables can be used to make active substances in vehicles such as water for injection, such as sesame oil, coconut oil, groundnut oil, cottonseed oil, naturally occurring vegetable oils, or synthetic resins such as oils. Dissolving or suspending agents are generally prescribed. Further, a buffering agent, a preservative, an antioxidant and the like may be mixed as necessary. For external preparations, petroleum jelly, paraffin wax, oils and fats, aqueous sheep fat, polyethylene glycol can be used as the base material, and ointments and creams can be prepared by ordinary methods. The food and beverage containing the cytotoxic toxin neutralizer of the present invention can be made into the above-mentioned preparation form, and can also be in the form of candy, scallop, biscuit, beverage, etc., and the required amount is added to each food raw material, and processed by a general manufacturing method. When ingested as a healthy food or functional food, it is used to prevent disease and maintain health. Therefore, it can be taken as a processed product that is taken orally several times a day and contains 5 mg to 5000 mg per day.

When adding a cytotoxic toxin neutralizer to these foods and drinks, the cytotoxic toxin neutralizer can be directly added in powder form, or the cytotoxic toxin neutralizer can be made into a 1 to 2% aqueous solution or ethanol solution or ethanol. The solution is added to the food and drink, 20 so that the final concentration is 1 to 10,000 PPm relative to the food and drink, and preferably 100 to 5000 ppm. The cavitation toxin neutralizing agent of the present invention can be used as a preventive agent when used for the purpose of preventing the digestive organ disease, and if used for the purpose of preventing the recurrence of the digestive organ disease that has been cured, then It can be used as a relapse prevention agent 14 200522973, and it can be used as a bactericide when it is used for the purpose of treating this digestive organ disease by removing Helicobacter pylori. Moreover, in the prevention, prevention of relapse, or treatment of the diseases of the digestive organs, the Helicobacter pylori bactericide of the present invention may be used alone, or 5 agents and / or antibiotics may be used in combination with proton pump.

The daily dose of one of the cytotoxic toxin neutralizers of the present invention can be appropriately selected according to its usage, patient age, gender and other conditions, the degree of disease, etc., usually the amount of the compound of the present invention as an active ingredient is 1 day per adult 0.1 to 2000 mg, preferably 0.5 to 1800 mg, and more preferably 1.0 to 1500 mg, can be divided into 1 to 4 10 times a day, for example, on an empty stomach. Examples are shown below, but the present invention is not limited to these examples. [Example 1] (By using a colloidal synthetic sorbent, a cytotoxic toxin neutralizing agent derived from Khubu flower capsules) 15 Crush 20g of Khubu flower blanket fruit in a bowl and stir with 2L of water In the

Extraction was carried out at 95 ° C for 40 minutes with stirring. After filtering, it was allowed to cool, and the extract was passed through a column filled with 80 ml of a hydrophilic ethylene polymer resin, and then washed with 400 ml of a 5% ethanol aqueous solution, and 400 ml of an 80% ethanol aqueous solution was passed through the same column. It was recovered and freeze-dried, and 800 mg of cavitation toxin neutralizing agent was obtained from a odorless and slightly bitter pale yellow 20-color powder. The yield of Congbu flower is 4%. [Example 2] (Modified by a colloidal synthetic sorbent derived from Khubu flower buds, a cytosolic toxin neutralizing agent) Stirring 20g of Khubu flower buds with 600ml of 50% ethanol solution, while stirring 15 200522973 at 30C 2 0 minute extraction. After filtration, the solution was concentrated under reduced pressure, and the concentrated solution was passed through a column filled with the present ethylbenzene and diethylbenzene resin, followed by washing / contesting with 400 claw 1 water, and passing 400 ml of 80% ethanol aqueous solution through. In the same column, 16 g of the cytotoxic toxin neutralizer 16 was obtained as a pale yellow powder with a odorless and slightly bitter taste, which was collected by cold / dong drying. The yield of Congbu cloth carpet is 8%. [Example 3] Said t; preparation of membrane-derived cavitation toxin neutralizing agent derived from Hubuhua blanket fruit) Yu Chuozhong crushed 20g of Hubuhuaweiguo and stirred it with 2L of water. The ® 10 is blended under 95. 〇Extract for 40 minutes. After filtering, it was allowed to cool. The ultrafiltration membrane with a molecular weight of 50,000 was isolated, and the extract was treated at room temperature of 10 kg / cm2 until it became 20 ml. The obtained residue was dried under reduced pressure, and 200 mg of a cavitation toxin neutralizing agent was obtained as a odorless, slightly bitter, light yellow powder. The yield of Congbu flower is 1%. 15 [Example 4] (Modified by an ultrafiltration membrane-derived cytotoxic toxin neutralizing agent derived from Khubu flower buds) Mix 600g of Khubu flower dad with 600ml of 50% ethanol aqueous solution, and stir under the stirring Extraction was performed at 80C for 40 minutes. After the extraction, the extracted solution was treated at 3.0 kg / cm2 and room temperature with an ultra-thin membrane of molecular weight I · until it became 20. The resulting residual liquid was cooled; dried in the east, and odorless with a slightly bitter taste-a yellow powder to obtain a cavitation toxin neutralizer 08g. The yield of Congbu flower was ㈣. [Example 5] (Further refinement and qualitative analysis of the cytotoxic toxin neutralizing agent) 0.8g of the cytotoxic toxin neutralizing agent obtained in Example 2 was dissolved in 500 ml of a 10% ethanol aqueous solution and borrowed. Xuanfen 1.0 kg / cm2, ultra-filtration membrane with 5,000 knives treated at room temperature, lyophilized into 20 liquids, and lyophilized, and it is slightly bitter without odor. until. The obtained residue neutralizer was 0.4 g. Chromatographic analysis was performed on the condition that the powdery dream powder and muscle-colored powder were obtained with cytotoxicity, as shown in Figure 3, and the conditions were determined by HPLC. The value of the general polyphenols was determined to obtain a value of 40.6%. When "° A determination method", it was converted into catechin (HPLC conditions). Device: Shimadzu LCM0A system, 10

.,, Brother & column: ODS-80TM (Dongyi, • 6mmI, D.x25cm), mobile phase: from You (A solution: B solution) = (100: 0) to " 5〇 · 5〇) 30-minute linear gradient, liquid A: 5% ethyl guess (including

Hcl), B liquid, acetonitrile, sample injection amount: 20 m, detection: detection of multiple wavelengths between 2000 and lion legs. [Example 6] (Preparation of a cavitation toxin neutralizing agent derived from unripe apples) After homogenizing 400 g of unripe apples (average weight 5.03 g) with 1% hydrochloric acid acid methanol, the mixture was heated and refluxed. Extract (3 times), concentrate the extract under reduced pressure and distill off the methanol, add chloroform for distribution (2 times), recover the water layer, and filter the distilled water to 200m. Use Sep-pak C18 20 by solid-phase extraction, and freeze-dried to obtain a cytotoxic toxin neutralizer. [Example 7] (bonding agent, capsule) The material obtained in Example 5 was 10.00 g milk bran 75.0 g 17 200522973 magnesium stearate 15.0 g total 100.Og The above weight parts were uniformly mixed, according to the general method Making lozenges and capsules. In addition, the substances obtained in Examples 1, 2, 3, 4, and 6 were added in place of the substances obtained in Example 5, respectively, and tablets and capsules were prepared in the same manner. [Example 8] 5 (powder, granule) 20.0g of the substance obtained in Example 5 starch 30.0g lactose 50.0g

A total of 100.Og The above parts by weight are uniformly mixed, and powders and granules are prepared according to a general method. In addition, the substances obtained in Examples 1, 2, 3, 4, and 6 were added in place of the substances obtained in Example 5, respectively, and tablets and capsules were prepared in the same manner. [Example 9] 10 (injection) The substance obtained in Example 5 l.og surfactant 9.0g physiological saline 90.0g total lOO.Og

Each of the above parts by weight is mixed with heat and sterilized to prepare an injection. In addition, the substances obtained in Example 1, 2, 3, 4, and 6 were added instead of the substances obtained in Example 5, respectively, and injections were prepared in the same manner. [Example 10] 15 (饴) 20.0g sucrose (75% solids) 70.0g water 9.5g coloring material 0.45g flavor 0.045g material obtained according to Example 5 0.005g 18 200522973 total lOO.Og using the above Each ingredient in each part by weight was prepared in accordance with a general method. In addition, the substances obtained in Example 1, 2, 3, 4, and 6 were added in place of the substances obtained in Example 5, respectively, and osmium was produced in the same manner. [Example 11] (Juice) Concentrated willow juice 15.0g Fructose 5.0g Citric acid 0.2g Spice 0.1g Pigment 0.15g Sodium ascorbate 0.048g According to Example 5, 0.002g water 79.5g total 100. Og

Juices are prepared using the above-mentioned components by weight of each component according to a general method. In addition, the substances obtained in Example 1, 2, 3, 4, and 6 were added in place of the substances obtained in Example 5, respectively, and fruit juice was produced in the same manner. [Example 12] 10 (biscuits)

Low-gluten flour 32.0g whole eggs 16.0g butter 16.0g granulated sugar 25.0g water 10.8g baking powder 0.298g 0.002g of the substance obtained in Example 5 total 100.Og The above-mentioned ingredients are used to make biscuits according to the general method. In addition, the substances obtained in Example 1, 2, 3, 4, and 6 were added in place of the substances obtained in Example 5, respectively, and biscuits were produced in the same manner. [Example 13] 19 200522973 Cytotoxicity test of vaccinated toxin on cultured cells AZ_521 cells from a human gastric cancer cell line or G401 cells from a human kidney cancer cell line were adjusted to a suspension of 20 × 10 5 ceUs / ml. After injecting the suspension 100 // 1 into 96-well dishes, a single 5-layer membrane for each cell was prepared overnight. In addition, a fixed concentration of the cytosolic toxin and various concentrations of the cytosolic toxin neutralizing agent obtained in Example 5 or 6 were added to the above 96-well dish after incubation for 37 minutes at 37x :. The final concentration of cavitation toxin was 120 nM ', and the final concentration of Example 5 or 6 was 0 to 100 # g / ml. After incubating the dish for 8 hours in 5% co2 ring and 37 C, the degree of neutral red (005% pBS 10 solution) was absorbed by the air cells (Ab540) to evaluate the effect of air cell toxins on the cells. Of toxicity. The results are shown in Figures 4 and 5. Depending on the concentration of the cytotoxic toxin neutralizing agent obtained in Examples 5 and 6, the cytotoxicity caused by the cytotoxic toxin to both AZ-521 cells and G40i cells was non-toxic. [Example 14] 15 Combination with cultured cells AZ-521 cells from a human gastric cancer cell line or G4G1 cells from a human kidney cancer cell line were adjusted to a suspension of 2 Gxl (5) cells / ml. After injecting this suspension liquid 100 // 1 into 96-well dishes, they were left overnight to prepare a single-layer membrane for each cell. In addition, the biomarker cavitation toxin of various concentrations and the cavitation toxin neutralizing agent obtained in Example 5 or 6 at a fixed concentration of 20 were added to the cell's monolayer membrane after listening and incubation for% minutes. The final concentration of cavitation toxin is 0 to 100, and the final concentration of Example 5 or 6 is-. The cells were plated at 5% CO2, 37 early. (: After 4 hours in culture, the cells were fixed with 025% glutaric acid. The biotin-labeled horseradish peroxizine (㈣ 脑 ㈤ 20 200522973 and the color development of TMBZ pigment (Ab450nm) were used to evaluate. The amount of the biomarker cytosolic toxin on the cell surface. The results are shown in Figures 6 and 7. Depending on the concentration of the cytosolic toxin neutralizing agent obtained in Examples 5 and 6, the binding of the cytosolic toxin to the cells is hindered. 5 [Example 15] Experiment of rat stomach injury

C57BL / 6J mice at 4 weeks of age who have been on a hunger strike (free access to drinking water only) for 24 hours, use oral probes to administer 5 // g of cytosolic toxin and 50 ~ 250 // g per 10g of body weight The emptying toxin neutralizing agent obtained in Example 5. Each of the 10 animal lines was bred in individual cages, and their stomachs were removed 48 hours after administration. The extracted specimens were fixed with 10% morpholine, and observed with a solid microscope before and after. Fixed specimens were stained with hematoxylin and eosin, and the degree of gastric injury was counted in accordance with the method of Ghiara et al. (Ghiara. P., et al. Infect. Immun. 63, 4154-4160. (1995)). Evaluation. The results are shown in Table 1. Example 5 can significantly suppress stomach damage. 【Table 1】

Number reagent Stomach injury scar 1 Phosphate buffer 1.6 ± 0.8 2 Example 5 (250 // g) 1 · 8 ± 0.8 3 Phytotoxin (5 // g) 3.0 ± 0 · 8 4 Phytotoxin (5 /// g) + Example 5 (50 // g) 2 · 4 ± 1 · 0 5 Phytocytosis toxin (5 // g) + Example 5 (100 // g) 2 · 2 ± 0 · 8 * 6 Phytocytosis Toxin (5 // g) + Example 5 (250 // g) 2.2 ± 0 · 8 * * It is a significant difference compared with 3% or less of the hazard rate. [Brief description of the figure] Figure 1 is a graph showing the analysis results of GPC (Collective Transmission Color Layer Analysis) from grape seed proanthocyanidins from Hubuhua. 20 Figure 2 is a graph showing the extinction scores of anthocyanins from grape seeds before the flowers of Houbu. 21 200522973 Fig. 3 is a graph showing the results of HPLC analysis of grape seed proanthocyanidins from Hubuhua. Fig. 4 is a graph showing the non-toxicity of cytoplasmic toxin in cultured cells of human gastric cancer cell AZ-521 (Example 13). Fig. 5 is a graph showing the non-toxicity of a cytosolic toxin in cultured cells of human kidney cancer cell G401 (Example 13).

Fig. 6 is a diagram showing that in the cultured cells of the human gastric cancer cell AZ-521, the access of the vacuolar toxin to the cells is prevented (Example 14). 10 Fig. 7 is a diagram showing that in the cultured cells of human kidney cancer cell G401, the access of the vacuolar toxin to the cells is impeded (Example 14). [Description of main component symbols] None

twenty two

Claims (1)

200522973 5 10 15 20 10. Scope of patent application: L 1 heavy grape seed proanthocyanidins have the effect of neutralizing (non-toxic) the cytotoxic toxin produced by H. pylori. 2 ‘μ 请 专 · 项 之 Grape # anthocyanins’, where the grapes express anthocyanins from the anthocyanins from the grapes of Hubuhua or Hubuhua dad. 3: The anthocyanins of grapevine before the application of the scope of patent application, which makes the grape express the anthocyanins of grapevine from the apple. -A prophylactic agent, a relapse preventive agent or a therapeutic agent for Helicobacter pylori-related gastrointestinal diseases, which contains grape seed proanthocyanidins as an active ingredient. —A kind of preventive agent, recurrence preventive agent or therapeutic agent for Helicobacter pylori-related digestive organ diseases, which contains grape seed proanthocyanidins as the active ingredient of the patent application Fan Bingwei 2 items. A prophylactic agent, a relapse preventive agent or a therapeutic agent for homogenizing helicobacter pylori-related digestive cry and homogenizing Yilu disease, which contains a patent application as an active ingredient. Chrysanthemum vinifera L. is an anthocyanin. 7.—A cavitating toxin neutralizing agent produced by Helicobacter pylori, which contains the proanthocyanidins from grape vines as the active ingredient. A cavitation toxin neutralizing agent produced by Helicobacter pylori is the active ingredient of grape seed proanthocyanidins which is claimed in item 2 of the patent. It is a kind of Helicobacter pylori produced by a natural neutralizing agent. It contains the grapevine Yu # 主 主 1 in the patent scope No. 3, as an active ingredient. 10. · A non-pharmaceutical product containing grape seeds with the right of ▲ Zhu Kui 3 and the first patent application scope: 4 5. 6 Kehua 23 200522973 green pigments as active ingredients. 11. A non-pharmaceutical product containing grape seed proanthocyanidins as the active ingredient in the second patent application. 12. — A kind of non-medicinal product, which contains grape seed proanthocyanidins 5 as the active ingredient in the patent application scope item 3. 13. A food and drink product containing grape seed proanthocyanidins as the active ingredient in the first patent application. 14. A food and drink product containing grape seed proanthocyanidins as an active ingredient in the second patent application. 10 15. A food and drink product containing grape seed proanthocyanidins as the active ingredient in the third patent application.